Zago Chaiene Evelin, Silva Sónia, Sanitá Paula Volpato, Barbugli Paula Aboud, Dias Carla Maria Improta, Lordello Virgínia Barreto, Vergani Carlos Eduardo
Department of Dental Materials and Prosthodontics, Araraquara Dental School, UNESP-Univ Estadual Paulista. Araraquara, São Paulo, Brazil.
IBB-Institute for Biotechnology and Bioengineering, Center of Biological Engineering, University of Minho, Braga, Portugal.
PLoS One. 2015 Apr 13;10(4):e0123206. doi: 10.1371/journal.pone.0123206. eCollection 2015.
Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.
多种微生物生物膜是一个研究不足但具有临床相关性的问题。本研究评估了白色念珠菌与甲氧西林敏感（MSSA）和耐药（MRSA）金黄色葡萄球菌在单物种和双物种生物膜中生长时的相互作用。通过互补方法评估单物种和双物种的黏附（90分钟）以及生物膜（12、24和48小时）：计算菌落形成单位（CFU/mL）、XTT还原法和结晶紫染色（CV）。还使用荧光试剂盒评估48小时生物膜水解酶的分泌情况。使用扫描电子显微镜（SEM）评估生物膜结构。定量分析结果使用双向方差分析和Tukey事后检验进行比较，而酶活性数据通过单向Welch方差分析，随后进行Games-Howell事后检验（α = 0.05）进行分析。白色念珠菌、MSSA和MRSA在单培养或混合培养中均能黏附并形成生物膜。一般来说，两种生长条件下的所有微生物随着时间推移细胞数量和代谢活性都逐渐增加，在12小时至48小时之间达到峰值（ρ<0.05）。白色念珠菌单生物膜和双生物膜的总生物量值显著高于细菌单生物膜（ρ<0.05）。除了MRSA单生物膜外，两种生长条件下的所有微生物都分泌蛋白酶和磷脂酶C。SEM图像显示细菌广泛黏附于白色念珠菌的菌丝成分。白色念珠菌、MSSA和MRSA可以在生物膜中共存而无拮抗作用，且存在明显的协同效应，细菌细胞优先与白色念珠菌的菌丝形式相关联。
